In 2009 we continued our accrual of patients into our blister study protocol. The protocol involves the formation of blisters on the forearms of human volunteers to study the inflammatory response in vivo. This year, we intiated a project comparing the transcriptomes of peripheral blood neutrophils with matched inflammatory exudate (blister) neutrophils from seven normal donors. For this project, we have teamed with the Rocky Mountain Laboraty Research Technologies Branch (RML) who will provide expert microarray analysis of these samples. We are increasing the number of chronic GvHD patients in our study pool (now a total of 4 patients) as they become available from our collaborators in Dr. Harry Malech's laboratory. Also, with the help of LCID, we have now studied a total of five patients with Job's syndrome and are awaiting quantitative analysis by our contractor at SAIC-Frederick. (Zarember 20% effort). In addition to several normals, we have also studied three patients with chronic graft-versus-host disease and two patients with Jobs Syndrome. Additional patients will be required to fully power these studies to determine whether the observed dysregulation of cytokine production is statistically significant (Kol Zarember, 20% effort). In 2009 we continued to study the molecular basis for priming and activation of the NADPH oxidase and superoxide production by endotoxin (LPS). The NADPH oxidase (NOX), an oligomeric enzyme, plays a key role in polymorphonuclear neutrophil (PMN)-mediated host defense by producing cytotoxic superoxide anion (O2.-). Whereas in vitro and biochemical studies have examined the assembly and activation of this important host immune defense system, few studies have examined the function of NOX in human patients with primary immunodeficiencies other than chronic granulomatous disease. We studied the activation of NOX in PMN from patients with two distinct immunodeficiencies, interleukin-1 receptor associated kinase 4 (IRAK4) deficiency and nuclear factor kappa (NF-&#954;B) essential modulator (NEMO or IKK&#947;) deficiency. We observed impaired O2.- generation by LPS-treated and fMLP-activated IRAK4-deficient PMN that correlated with decreased phosphorylation of p47phox and subnormal translocation of p47phox, p67phox, Rac2, and gp91phox/Nox2 to the membranes indicating that TLR4 signaling to the NOX activation pathway requires IRAK4. NEMO-deficient PMN also generated less O2.- in response to LPS and fMLP and translocated less p47phox and p67phox to membranes than normal PMN but were more responsive than IRAK4-deficient cells. Decreased LPS and fMLP induced phosphorylation of p38 MAPK in both IRAK4- or NEMO-deficient PMN and of p21-activated kinases (PAK) in IRAK4-deficiency implicates additional signal transduction pathways in regulating PMN superoxide activation by LPS and fMLP (Anjali Singh, 85% effort;Kol Zarember 10% effort). In 2009 we initiatied a study of granuloma formation utilizing immunofluorescence to probe the various cellular and molecular components of granulomas. Using this technique, we will demonstrate the various types of immune cells (including neutrophils, monocytes, T and B cells, and fibroblasts) and immune mediators (such as IL-1, IL-8, IL-10, TGF, and various antimicrobial peptides) present in granulomas and potentially contributing to their formation. We have validated the antibodies against many of these targets in peripheral blood smears, a formalin fixed, paraffin embedded buffy coat pellet, and normal formalin fixed, paraffin embedded spleen and bone marrow sections to ensure that the desired binding is achieved with minimal background/non-specific binding. Experiments are underway using liver, spleen, and lung tissue from patients with CGD that contain granulomas and efforts are being made to obtain granuloma-containing tissue from patients with other diseases such as inflammatory bowel disease and sarcoidosis. (Soule, 30% effort). This past year we initiated another project to better understand the potential of microRNA in the regulation of phagocyte function, including regulation of production of cytokines. MicroRNAs comprise a class of over 500 small, non-coding RNA that regulate gene expression by inhibiting RNA translation. They are felt to regulate the expression of over 30% of the genome and specific microRNA species have been shown to be active in neutrophils. A complete survey of the microRNA species in neutrophils and monocytes has not been performed. We have used several commercially available RT-PCR kits to assay specific microRNA in neutrophils, however this has been complicated by significant experiment to experiment variation. In response to this, we have been collaborating with the Rocky Mountain Laboratory to perform whole RNA sequencing on total RNA isolated from purified neutrophils preparations from normal controls and patients with both X-linked (gp91-deficient) and autosomal recessive (p47-deficient) CGD patients. This information will itself be novel, but will also facilitate the development of future studies involving both microRNA microarrays and conventional RT-PCR. We have also begun preliminary work studying the viability of transfecting microRNA precursors and inhibitors into neutrophils to determine the effect of altering microRNA expression on neutrophil function. Finally, we have been speaking with other investigators about the possibility of studying the role of microRNA in myeloid stem cells (CD34+ cells). There is a large body of literature demonstrating the role of microRNA in development at the cellular and organism level. It is likely that microRNA are active in directing myeloid stem cell development and differentiation, but this has not been extensively studied.